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Title Regulation of STAT3-mediated signaling by LMW-DSP2.

Author(s) Sekine, Y.; Tsuji, S.; Ikeda, O.; Sato, N.; Aoki, N.; Aoyama, K.; Sugiyama, K.; Matsuda, T.

Citation Oncogene, 25(42): 5801-5806

Issue Date 2006-09-21

Doc URL http://hdl.handle.net/2115/22102

Rights Nature Publishing Group, ONCOGENE, 25, 42, 2006, 5801-5806.

Type article (author version)

File Information ONCO25-42.pdf

Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP

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Title: Regulation of STAT3-mediated signaling by LMW-DSP2

Authors: Yuichi Sekine1, Satoshi Tsuji1, Osamu Ikeda1, Noriko Sato1, Naohito Aoki2,

Koji, Aoyama3, Kenji Sugiyama4 and Tadashi Matsuda1, *

Affiliation: 1Department of Immunology, Graduate School of Pharmaceutical Sciences

Hokkaido University, Sapporo 060-0812 Japan, 2Laboratory of Molecular Food

Chemistry and Biochemistry, Department of Life Sciences, Faculty of Bioresources,

Mie University, 1577 Kuriyamachiya-cho, Tsu, Mie, 514-8507 Japan, 3Department of

Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya

University, Furo-Cho, Chikusa-Ku, Nagoya 464-8601, Japan, 4Nippon Boehringer

Ingelheim Co., Ltd., Kawanishi Pharma Research Institute, 3-10-1 Yato, Kawanishi,

Hyogo 666-0193, Japan.

*Address for manuscript correspondence: Dr. Tadashi Matsuda, Department of

Immunology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-

Ku Kita 12 Nishi 6, Sapporo 060-0812, Japan TEL: 81-11-706-3243, FAX: 81-11-706-

4990, E-mail: tmatsuda@pharm.hokudai.ac.jp

Running title: Regulation of STAT3-mediated signaling by LMW-DSP2

Keywords:IL-6; LIF; STAT3; phosphatase; transcriptional regulation

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Abstract

Signal transducer and activator of transcription 3 (STAT3), which mediates

biological actions in many physiological processes, is activated by cytokines and

growth factors, and has been reported to be constitutively activated in numerous

cancer cells. In this study, we examined whether low molecular weight-dual

specificity phosphatase two (LMW-DSP2) is involved in the regulation of the

interleukin 6 (IL-6)-/leukemia inhibitory factor (LIF)-/STAT3-mediated signaling

pathway. IL-6/LIF induced LMW-DSP2 expression in murine testicular or

hepatoma cell lines, while LMW-DSP2 overexpression in 293T cells suppressed IL-

6-induced phosphorylation and activation of STAT3. Furthermore, LMW-DSP2

suppressed the expression of IL-6-induced endogenous genes. In contrast, small-

interfering RNA-mediated reduction of LMW-DSP2 expression enhanced IL-6-

induced STAT3-dependent transcription. In fact, LMW-DSP2 interacted with

STAT3 in vivo and endogenous LMW-DSP2 bound to STAT3 in murine testicular

GC-1 cells. These results strongly suggest that LMW-DSP2 acts as a negative

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regulator of the IL-6/LIF/STAT3-mediated signaling pathway.

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The signal transducer and activator of transcription (STAT) is known to mediate cell

proliferation, differentiation and survival in immune responses, hematopoiesis,

neurogenesis and other biological processes (Darnell et al.,1994; Ihle,1996;

O’Shea,1997). For example, STAT3 is involved in the epithelial-mesenchymal transition

during gastrulation, organogenesis, wound healing and cancer progression (Levy et

al.,2002). Constitutive or dysregulated expression of STATs has been identified in

cancer cells and oncogene-transfected cells and also shown to be involved in a wide

range of other diseases, including autoimmune diseases (Levy et al.,2002;Bromberg et

al.,2000). Therefore, STAT activation is tightly regulated by a variety of mechanisms.

The protein inhibitor of activated STAT (PIAS) family of proteins decreases STAT-

dependent transcription by blocking STAT-DNA binding in the nucleus (Shuai et

al.,2003). Suppressor of cytokine signaling (SOCS) proteins are induced by STATs and

play roles in the negative feedback of STAT activation (Yasukawa et al.,2000).

Cytoplasmic tyrosine phosphatases, such as SH2-containing phosphatase 1 (SHP1),

SHP2 and protein-tyrosine phosphatase 1B (PTP1B), also prevent further STAT

activation in the cytoplasm (Shuai et al.,2003; Yasukawa et al.,2000). Nuclear tyrosine

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phosphatases, such as TC45, dephosphorylate nuclear STATs, thereby allowing them to

return to the cytoplasm (Shuai et al.,2003). We further reported that the nuclear isoform

of TC-PTP was a potential negative regulator of interleukin 6 (IL-6)-mediated signaling,

through STAT3 dephosphorylation and deactivation, as well as prolactin/STAT5-

mediated signaling (Aoki et al.,2002;Yamamoto et al.,2002).

Dual specificity phosphatases (DSPs)/MAP kinase phosphatases (MKPs) are known

to regulate MAP kinase-mediated signaling pathways, including ERK, JNK or p38

MAPK (Alonso et al.,2003). In previous studies, we cloned a distinct class of low

molecular weight DSPs (LMW-DSPs) (Aoyama et al.,2001;Aoki et al.,2001) that

contain a single catalytic domain, but lack a putative common docking site for MAPKs,

designated the cdc25 homology domain. The first LMW-DSP to be cloned was the VH1

protein from the Vaccinia virus (Guan et al.,1991). A related phosphatase was cloned in

mammalian cells and designated VHR, for VH1-related (Ishibashi et al.,1992). Both

VH1 and VHR differ from other DSPs in that they are much smaller (19 and 21 kDa,

respectively). VH1 has been reported to dephosphorylate both MAP kinases and STAT1

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(Najarro et al.,2001), while VHR appears to be specific for ERK and JNK (Denu et

al.,1995;Alonso et al.,2001). LMW-DSP2 was found to dephosphorylate and deactivate

p38 MAPK and JNK, but not ERK (Aoyama et al.,2001). LMW-DSP2 has also been

referred to as VHX (Alonso et al.,2002), JSP1 (Shen et al.,2001) and JKAP (Chen et

al.,2002). However, the physiological functions of LMW-DSPs have remained unclear,

since they appear to be less efficient than many other MAPK-specific DSPs.

Dephosphorylation of activated STATs is one of the key regulatory mechanisms in

cytokine signaling. In a previous study, we showed that the nuclear isoform of TC-PTP

dephosphorylated PRL-activated STAT5a and STAT5b and IL-6/leukemia inhibitory

factor (LIF)-activated STAT3 (Aoki et al.,2002;Yamamoto et al.,2002). As mentioned

above, we also cloned a new class of DSPs (LMW-DSP-1, -DSP2, -DSP4, -DSP6, -

DSP10 and -DSP11) from a mouse testis cDNA library and found that they were

specifically and abundantly expressed in the testes (Aoyama et al.,2001;Aoki et

al.,2001). Recently, LMW-DSP2 was found to belong to the subfamily of small DSPs

related to the Vaccinia virus VH1 phosphatase (Aoyama et al.,2001;Guan et

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al.,1991;Alonso et al.,2002;Shen et al.,2001;Chen et al.,2002). The finding that VH1

phosphatase blocked interferon (IFN)-g signaling by dephosphorylating STAT1 (Najarro

et al.,2001) led us to examine whether LMW-DSP2 is involved in the regulation of the

STAT3-mediated signaling pathway.

To investigate the involvement of LMW-DSP2 in STAT3-mediated signaling, we first

examined whether LMW-DSP2 expression was regulated by the IL-6 family of

cytokines in the mouse testicular cell line GC-1 and mouse hepatoma cell line Hepa 1-6

using RT-PCR. As shown in Figure 1a (left panel), immediate early induction of LMW-

DSP2 mRNA expression (at 15 min) was observed in GC-1 cells after treatment with

LIF. Furthermore, IL-6 stimulated LMW-DSP2 mRNA expression in Hepa 1-6 cells at

an immediate early time point, similar to the case with LIF (Figure 1b; left panel). We

could also observe the LIF/IL-6/STAT3-mediated SOCS3 mRNA expression (at 30

min) in these cells (Figure 1a and b; left panels). When we monitored STAT3

phosphorylation in aliquots of these cell extracts after similar treatments with LIF or IL-

6, LIF and IL-6 each stimulated phosphorylation of Tyr705 in STAT3 at 15 min after

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stimulation in both cell types (Figure 1a and b; right panels). However, neither LIF nor

IL-6 treatment showed any significant induction of Ser727 phosphorylation in STAT3.

We attempted to examine the changes of protein level of LMW-DSP2. Unfortunately,

we could not detect the endogenouos protein of LMW-DSP2 in the total lysates of these

cells, because of the low detection sensitivity of anti-LMW-DSP2 antibody

immunoblotting. We also tested whether very early induction of LMW-DSP2 by IL-6 or

LIF is dependent on the ERK pathway using an ERK inhibitor, U0126. Treatment of

U0126 in GC-1 cells resulted in a reduction of IL-6 or LIF-induced LMW-DSP2

expression (data not shown). These results indicate that LMW-DSP2 mRNA expression

is induced by LIF or IL-6 in mouse testicular and hepatoma cells through the ERK

pathway.

To examine whether LMW-DSP2 has any effects on STAT3-mediated transcriptional

activation, we used transient transfection assays. The STAT3-mediated transcriptional

responses were measured by using STAT3-LUC, in which the a2-macroglobulin

promoter drives expression of a luciferase (LUC) reporter gene (Nakajima et al.,1996).

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293T cells transfected with STAT3-LUC were treated with LIF, and the LUC activities

were determined. When cells were co-transfected with LMW-DSP2, the transcriptional

activation of STAT3-LUC decreased in a dose-dependent manner compared with that of

mock vector-transfected cells (Figure 2a). Two amino acids in LMW-DSP2, namely Asp

(D)-57 and Cys (C)-88, have previously been shown to participate in the catalytic

mechanism of DSP activity (Aoyama et al., 2001). Wild-type (WT) as well as

catalytically inactive Asp/Ala (D/A) and Cys/Ser (C/S) forms of LMW-DSP2 were also

co-transfected into 293T cells. No decreases in STAT3 activation were observed when

the cells were co-transfected with LMW-DSP D/A and C/S (Figure 2b), suggesting that

the phosphatase activity of LMW-DSP2 is essential for STAT3 deactivation.

We further examined whether LMW-DSP2 acts as an inhibitor of IL-6/STAT3-

mediated transcriptional activation. To examine the effect of LMW-DSP2 on IL-6-

mediated transcriptional activation through STAT3, we performed transient transcription

assays using Hep3B and HeLa cells. The IL-6-mediated STAT3 transcriptional

responses were measured by STAT3-LUC, as described above. As shown in Figure 2c

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and d, expression of LMW-DSP2 WT, but not LMW-DSP2 D/A, deactivated IL-6-

induced transcriptional activation of STAT3-LUC in a dose-dependent manner in both

Hep3B and HeLa cells. In HeLa cells, IL-6 treatment induced STAT3-mediated

endogenous SOCS3 mRNA expression (Figure 2e). Next, we tested the effect of LMW-

DSP2 on the endogenous SOCS3 mRNA expression induced by IL-6. As shown in

Figure 2e, RT-PCR analyses revealed that IL-6-induced endogenous SOCS3 mRNA

expression was decreased in HeLa cells transfected with LMW-DSP2, but not a mock

vector. These results suggest that LMW-DSP2 acts as an inhibitor of IL-6-induced

transcriptional activation of STAT3 in Hep3B and HeLa cells.

To further explore whether LMW-DSP2 regulates STAT3-mediated transcriptional

activation, we used small interfering RNA (siRNA) to reduce the endogenous

expression of LMW-DSP2 in HeLa cells. HeLa cells were transfected with a specific

siRNA for LMW-DSP2 or a control siRNA, and aliquots of total RNA isolated from the

transfected cells were subjected to RT-PCR analysis, which confirmed reductions in

LMW-DSP2 mRNA expression. Next, we determined the effects of these siRNAs on

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IL-6-induced STAT3-LUC activation in HeLa cells. As shown in Figure 2f and g,

siRNA-mediated reduced expression of LMW-DSP2 resulted in a significant

enhancement of IL-6-induced STAT3-LUC activation and IL-6-induced SOCS3 mRNA

expression, strongly suggesting that LMW-DSP2 regulates STAT3-mediated

transcriptional activation in HeLa cells. Similarly, we examined the effect of LMW-

DSP2 siRNA on interferon- or erythoropoietin-induced STAT activation in HeLa cells

(data not shown). However, we could not observe any significant enhancement of these

cytokine signaling, suggesting that LMW-DSP2 specifically acts STAT3-mediated

signaling.

Next, we assessed the changes in STAT3 phosphorylation, which triggers its

activation, in 293T cells. LMW-DSP2 WT or D/A was co-transfected with Myc-tagged

STAT3 into 293T cells. Time course analyses of LIF-induced STAT3 phosphorylation

demonstrated that, upon co-expression of LMW-DSP2 WT, ligand-induced tyrosine- or

serine-phosphorylation of STAT3 was remarkably decreased, compared with the levels

in mock vector- and LMW-DSP2 D/A-transfected cells (Figure 3a). We next examined

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LMW-DSP2 siRNA on IL-6-induced phosphorylation of STAT3 in HeLa cell. As

shown in Fig. 3b LMW-DSP2 siRNA treatment significantly enhanced IL-6-induced

phosphorylation of STAT3. These results indicate that LMW-DSP2 dephosphorylates

STAT3 and negatively regulates LIF-mediated STAT3 transcriptional activation in

293T cells. A STAT3 phosphatase, TC-PTP was also shown to dephosphorylate Jak

kinases to regulate cytokine signaling (Simoncic et al.,2002). Indeed, we could observe

dephosphorylation of tyrosine-phosphorylated Jak2 by overexpression of LMW-DSP2

in 293T cells (data not shown), suggesting that LMW-DSP2 regulates IL-6/LIF-

mediated signaling through dephosphorylation of Jaks and STAT3.

We further assessed the effect of LMW-DSP2 on the nuclear translocation of STAT3.

Expression vectors for FLAG-tagged STAT3 and/or Myc-tagged LMW-DSP-2 WT or

D/A were transfected into Hep3B cells. At 48 h after the transfection, the cells were left

untreated or treated with IL-6. As shown in Figure 3b (right panels), STAT3 translocated

into the nucleus of Hep3B cells after 30 min of stimulation with IL-6. In a previous

study, we used indirect immunofluorescence staining to show that LMW-DSP2 was

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localized throughout the cytoplasm and nucleus in COS7 cells (Aoyama et al.,2001). In

Hep3B cells, LMW-DSP2 was also localized throughout the cytoplasm and nucleus, but

was predominantly present in the cytoplasm. Next, we examined the co-localization of

LMW-DSP2 with STAT3 in Hep3B cells. As shown in Figure 3b, STAT3 failed to

translocate into the nucleus when co-transfected with LMW-DSP2 WT, but not LMW-

DSP2 D/A, although the staining pattern of the translocated STAT3 tended to be diffuse,

even after co-transfection with LMW-DSP2 D/A. Therefore, overexpression of LMW-

DSP2 inhibits the nuclear translocation of STAT3 mainly through dephosphorylation of

STAT3. We also tested the effect of siRNA on STAT3 nuclear translocation. However,

we could not detect a significant enhanced nuclear accumulation of STAT3 by siRNA

treatment.

One of the mechanisms consistent with the above-described data is a direct

interaction between STAT3 and LMW-DSP2, which triggers its deactivation. We first

tested this possibility by co-immunoprecipitation experiments. Expression vectors

encoding Myc-tagged LMW-DSP2 WT together with or without FLAG-tagged wild-

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type STAT3 (STAT3 WT) or STAT3 YF, which has a substitution of Tyr(Y)-705 for Phe

(F) (Nakajima et al.,1996) were transiently transfected into 293T cells. At 36 h after

transfection, the transfected 293T cells were stimulated with or without LIF and then

lysed, immunoprecipitated with an anti-FLAG antibody. The immunoprecipitates

obtained were analyzed by western blotting with an anti-Myc antibody. As shown in

Figure 4a, LMW-DSP2 bound to STAT3WT but not to STAT3 YF, suggesting this

interaction occurs in a phosphotyrosine-dependent manner. We next tested the

interaction of STAT3 with a series of LMW-DSP2 mutants. Expression vectors

encoding FLAG-tagged STAT3 and Myc-tagged LMW-DSP2 WT or its inactive

mutants D/A or C/S were transiently transfected into 293T cells. As shown in Figure 4b,

STAT3 interacted with each of LMW-DSP2 WT, D/A and C/S. Although LMW-DSP2

C/S showed a slightly stronger interaction with STAT3 than LMW-DSP2 WT, LMW-

DSP2 D/A showed a weaker interaction with STAT3 than both LMW-DSP2 WT and

C/S, suggesting that Asp-57 may be close to the binding site of STAT3 on LMW-DSP2.

To further confirm that endogenous LMW-DSP2 interacts with STAT3 in vivo, cell

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extracts obtained from LIF-stimulated GC-1 cells were subjected to co-

immunoprecipitation experiments. As shown in Figure 4c, anti-LMW-DSP2

immunoprecipitates of GC-1 cell extracts contained the STAT3 protein. This result

suggests that endogenous LMW-DSP2 interacts and forms a complex with STAT3 in

GC-1 cells.

In this study, for the first time, we have demonstrated that LMW-DSP2 is an

important regulator of STAT3 functions in the downstream of IL-6/LIF signaling, and

may thus play critical roles in the progression of IL-6-related diseases. More detailed

understanding of the interaction between STAT3 and LMW-DSP2 is therefore important,

since this new information may allow the development of novel therapeutic approaches

for these conditions.

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Acknowledgement

This work was supported in part by grants from the Osaka Foundation for Promotion of

Clinical Immunology, the Naito Foundation, the Akiyama Foundation and the Sasakawa

Scientific Research Grant from The Japan Science Society.

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Titles and legends for figures

Figure 1. LIF and IL-6 induced mRNA expression of LMW-DSP2 in testicular and

hepatoma cells

Murine testicular cell line, GC-1(a) and hepatoma cell line, Hepa 1-6 (b), were

maintained in DMEM containing 10% fetal calf serum (FCS). Cells were treated or

untreated with LIF (INTERGEN) (100 ng/ml) or IL-6 (a kind gift from Ajinomoto Co.)

(50 ng/ml) for the indicated periods. Total RNA samples isolated from these cells were

subjected to RT-PCR analysis using LMW-DSP2, SOCS3 and glyceraldehydes-3-

phosphate dehydrogenase (G3PDH) primers (Aoyama et al.,2001). RT-PCR products

were separated on a 1% agarose gel. After the similar treatment with LIF or IL-6, the

cells were then lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, containing

1% NP-40, 1 mM sodium orthovanadate and 1 mM phenylmethylsulfonyl), and an

aliquot of total extracts were examined with Western blot using anti-pSTAT3 (Tyr705),

anti-pSTAT3 (Ser727) (Cell signaling Technologies) or anti-STAT3 antibody (Santa

Cruz). This figure is representative of three separate experiments.

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Figure 2. LMW-DSP2 deactivated LIF- or IL-6-induced STAT3-mediated

transcriptional activation

a. Human embryonic kidney carcinoma cell line, 293T, was maintained in DMEM

containing 10% FCS and transfected in a 12-well plate were transfected with STAT3-

LUC (Nakajima et al.,1996) (0.4 mg) and/or indicated amounts of empty vector or

expression vector for LMW-DSP2 by the standard calcium precipitation protocol. At 36

h after transfection, the cells were stimulated with LIF (100 ng/ml) for additional 8 h.

The stimulated cells were harvested, and luciferase activities were measured (Sekine et

al.,2005). b. 293T cells in a 12-well plate were transfected with STAT3-LUC (0.4 mg)

and/or indicated amounts of WT, D/A or C/S of LMW-DSP2 as described the above. c

and d. Human hepatoma cell line Hep3B and human cervix carcinoma cell line, HeLa,

were maintained in DMEM containing 10 % FCS (Imoto et al.,2003;Muromoto et

al2004). Hep3B(c) and HeLa (d) cells in a 12-well plate were transfected with STAT3-

LUC (0.5 mg) and/or indicated amounts of WT or D/A of LMW-DSP2 using jetPEI

(PolyPlus-transfection). At 36 h after transfection, the cells were stimulated with IL-6

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(50 ng/ml) for additional 6 h. The stimulated cells were harvested and assayed for the

luciferase activity using the Dual-Luciferase Reporter Assay System (Promega). At least

three independent experiments were carried out for each assay. e. HeLa cells in a 6-well

plate were transfected with empty vector or Myc-LMW-DSP2 WT (0.5 mg). At 36 h

after transfection, the cells were stimulated with or without IL-6 (50 ng/ml) for the

indicated periods. Total RNA samples isolated from these cells were subjected to RT-

PCR analysis using SOCS3, G3PDH. RT-PCR products were separated on a 1% agarose

gel. LMW-DSP2 protein expression level was monitored by Western blot using anti-

Myc antibody. This figure is representative of three separate experiments. f. HeLa cells

in a 24-well plate were transfected with siRNA targeting human LMW-DSP2 using

Lipofectamine2000 (Invitrogen). siRNAs targeting human LMW-DSP2 used in this

study were as follows: 5'-CUCAAAACCUGACAAGACAUUUCAA-3'. The cells were

then transfected with STAT3-LUC using jetPEI (PolyPlus-transfection). At 36 h after

transfection, the cells were treated with IL-6 (50 ng/ml) for an additional 6 h. Total

RNAs from these cells which treated with IL-6 (50ng/ml) for 30 min were also analyzed

by RT-PCR using LMW-DSP2 or G3PDH primers, verifying siRNA-mediated reduction

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in endogenous LMW-DSP2. The cells were then harvested, and luciferase activities

were measured. The results are indicated as fold induction of luciferase activity from

triplicate experiments, and the error bars represent the S.D. g. HeLa cells were treated

with control siRNA or LMW-DSP2 siRNA as described the above and cells were

stimulated with IL-6 (50 ng/ml) for 30 min. Total RNA samples isolated from these

cells were subjected to RT-PCR analysis as described the above.

Figure 3. LMW-DSP2 inhibits LIF-induced tyrosine-phosphorylation and nuclear

translocation of STAT3

a. 293T cells in a 6 well-plate were transfected with Myc-tagged STAT3 (2 mg) together

with empty vector, Myc-tagged LMW-DSP2 WT or D/A mutant (4 mg). Forty-eight

hours after transfection, cells were starved for 3 h, followed by treatment with or

without LIF (100 ng/ml) for the indicated periods. The cells were lysed, and then

immunoblotted with anti-pSTAT3 (Tyr705) (upper panel), anti-pSTAT3 (Ser727)

(middle panel) or anti-Myc antibody (lower panel). b. HeLa cells were treated with

control siRNA or LMW-DSP2 siRNA as described the above and cells were stimulated

26

with IL-6 (50 ng/ml) for 30 min. The cells were lysed, and then immunoblotted with

anti-pSTAT3 (Tyr705) (upper panel) or anti-STAT3 antibody (lower panel). c. Hep3B

cells were transfected with FLAG-tagged STAT3 (1 mg). At 30 min after stimulation, the

cells were fixed with a solution containing 4% paraformaldehyde and reacted with

rabbit anti-FLAG antibody and/or mouse anti-Myc antibody. Hep3B cells were also

transfected with FLAG-tagged STAT3 (1 mg) together with Myc-tagged LMW-DSP2

WT or D/A mutant (1 mg). At 36 h after transfection, cells were treated with or without

IL-6 (50 ng/ml) for 30 min, and then fixed and reacted with anti-FLAG polyclonal

antibody and anti-Myc monoclonal antibody, and visualized with fluorescein

isothiocyanate (FITC)-conjugated anti-rabbit IgG and/or rhodamine-conjugated anti-

mouse IgG (Chemicon) and observed under a confocal laser fluorescent microscope

(Sekine et al.,2005). At the same time, the nuclei in the cells were stained with 4', 6-

diamidino-2-phenylindole (DAPI) (Wako). Images were obtained by using a Zeiss LSM

510 laser scanning microscope with an Apochromat x63/1.4 oil immersion objective

and x4 zoom.

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Figure 4. STAT3 and LMW-DSP2 physically interact in vivo

a. 293T cells (1x107 cells) were transfected with FLAG-tagged STAT3 WT (7.5 mg) or

STAT3 YF mutant (10 mg) together with or without WT of LMW-DSP2 (10 mg). At 36 h

after transfection, the cells were stimulated with LIF (100 ng/ml) for additional 8 h. The

cells were then lysed, and immunoprecipitated with anti-FLAG antibody and

immnoblotted with anti-Myc (upper panel) or anti-FLAG antibody (middle panel). Total

cell lysates (1%) were blotted with anti-pSTAT3 (Tyr705) (lower panel) or anti-Myc

antibody (bottom panel).

b. 293T cells (1x107 cells) were transfected with FLAG-tagged STAT3 (7.5 mg) together

with or without WT, D/A or C/S of LMW-DSP2 (10 mg). At 48 h after transfection, the

cells were lysed, and immunoprecipitated with anti-FLAG antibody and immnoblotted

with anti-Myc (upper panel) or anti-FLAG antibody (middle panel). Total cell lysates

(1%) were blotted with anti-Myc (bottom panel).

c. Murine testicular GC-1 cells (2x107) were stimulated with LIF for 30min and the cells

were lysed, and immunoprecipitated with control IgG or anti-LMW-DSP2 antibody and

immnoblotted with anti-STAT3 antibody (upper panels) or anti-LMW-DSP2 antibody

28

(lower panels).